The Epstein-Barr virus (EBV) is a virus of the Herpesviridae family which infects 90 to 95% of the world population. EBV, a virus with enveloped double-stranded DNA, preferentially infects the B lymphocytes but is also capable of infecting other cell lines, in particular the T lymphocytes, the macrophages and the epithelial cells. EBV enters the B lymphocytes by means of three essential glycoproteins (gp350/220, gp85 and gp42) allowing it to bind to the CD21 receptor and the molecules of the HMC (Histocompatibility Major Complex) of class II present on the surface of the quiescent B lymphocytes. The primary infection with EBV leads to an activation of the immune system of the host which then presents a standard serological profile, in particular by the production of antibodies of IgG and/or IgM isotypes directed against the viral antigens in particular EBNA (Epstein-Barr Nuclear Antigen), EA (Early Antigen) or VCA (Virus Capsid Antigen). This primary infection, which is generally asymptomatic, can lead to an infectious mononucleosis which resolves itself spontaneously in a few weeks in an immunocompetent patient.
Once the primary infection episode is resolved, EBV enters a latency stage. The EBV genome is circularized and is found again in episomal form in the nucleus of the infected cells. A very restricted number of viral genes is then expressed, which allow the virus not only to maintain its genome during division of the host cell but also to escape the vigilance of the immune system. Among these genes, those encoding the nuclear proteins EBNA-1 (Epstein-Barr Nuclear Antigen-1) required for the episomal replication and the maintenance of the viral genome, EBNA-2 (essential for the B lymphocyte immortalization process), EBNA-3 (involved in the establishment of the latent form of EBV), EBNA-5 (involved in the viral transformation), and the viral membrane proteins LMP-1 (Latent Membrane Protein-1) and LMP-2 play a major role. The non-coding viral RNAs EBER-1 (Epstein-Barr virus Encoded RNA-1) and EBER-2 also perform a key function by preventing the apoptosis of the host cells. In reality several latent forms exist, which correspond to the distinct expression profiles of the aforementioned EBV genes. Form 0, where no EBV gene is expressed, corresponds to the latent form of EBV found in the memory B lymphocytes once the primary infection is resolved; this is the least immunogenic form of EBV which thus allows the virus to completely escape the immune system throughout the life of the patient. Some of these memory B lymphocytes can also contain the latent form I (EBNA-1+/EBERs+) of EBV. The cells infected with the form II-A (EBNA-1+/EBNA-2−/LMP-1+) are found in the germinal centres of the amygdalae of healthy carriers. The latent form II-B (EBNA-1+/EBNA-2+/LMP-1−) corresponds to that found in the cells of the amygdalae of carriers suffering from infectious mononucleosis. The latent form III (expression of six EBNA proteins and three LMP proteins) constitutes the most immunogenic latent form of EBV and is generally found in the transformed cells in immunodeficient patients. This latent form of EBV is also considered as the most aggressive because it constitutes the only one to be able to effectively transform B lymphocytes in vitro.
The involvement of the different latent forms of EBV has been reported in numerous neoplastic-type pathologies (Burkitt's lymphoma, Hodgkin's and non-Hodgkin's lymphoma, nasopharyngeal carcinoma and gastric carcinoma, lymphoproliferative syndromes) not only in immunosuppressed or immunodeficient patients, but also in patients having an intact immune system (Carbon et al., The Oncologist 2008, 13: 577-585). However, immunocompetent individuals keep a latent viral infection under control (periodic reactivations of EBV well contained by the immune system), while immunosuppressed or immunodeficient individuals suffer viral reactivations capable of developing into malignant lymphoproliferations.
Lymphoproliferations represent a frequent complication which is most often lethal in patients with immune deficiency, whether of genetic origin (Wiskott-Aldrich's syndrome, X-linked lymphoproliferative syndrome), caused by an infection with the human immunodeficiency virus (HIV) or linked to immunosuppressive treatments in the case of transplant patients. For this last category of patients, the chronic antigenic stimulation linked to a transplant, the oncogenic effect of immunosuppressive treatments, the reduction in immune responses and EBV infection are among the factors that contribute to the development of such lymphoproliferation.
The post-transplant lymphoproliferative diseases (PTLDs) constitute a complication which is often fatal in patients having received a solid organ transplant or a transplant of haematopoietic stem cells. In patients having received a bone marrow transplant, PTLDs occur in the 12 months following the implantation of the transplant, i.e. before the T system is completely restored. Patients who have received transplants can develop PTLDs during the first year following the transplant (when the immunosuppressive treatment is most intense) but also during an episode of chronic prolonged immunosuppression (Ethel Cesarman, Cancer Letters 2011, 305(2): 163-172). PTLDs are characterized by uncontrolled proliferation of the B lymphocytes infected with EBV. In these immunosuppressed patients, hyporeactivity of the T lymphocytes promotes a polyclonal then monoclonal proliferation of B lymphocytes leading to malignancy.
The aforementioned pathologies develop as a result of EBV reactivation. This reactivation corresponds to the activation of the overall replication of EBV involving not only the latent origin of replication (oriP, which allows it to remain in the episomal form) but also the lytic origin of replication (oriLyt) of the virus. While the activation of oriP is carried out by the proteins of cellular origin associated with EBNA-1, that of oriLyt requires the intervention of several proteins of viral origin (Fixman et al., 1995, 69(5): 2998-3006). The activation of oriLyt leads to several consecutive cycles of replication of the viral DNA. EBV reactivation is characterized by strong replication involving both origins of replication of the virus.
During EBV reactivation, a minority of B lymphocytes infected with EBV in its latent form enter a lytic infection phase. During this phase, all of the viral proteins of EBV are produced, allowing the assembly of complete virions which lyse their host cells in order to then infect neighbouring cells (lytic cycle). The purpose of this horizontal transmission of EBV is to increase the pool of B lymphocytes infected with the EBV virus. In a lymphoproliferative context, the cells infected with the lytic form of EBV contribute to the process of tumour progression leading to lymphomas (Hong et al., J. Virology 2005, 79(22): 13993-14003/Ma et al., J. Virology 2011, 85(1): 165-177). These viral reactivations generally have no consequences in immunocompetent patients but can lead to malignant lymphoproliferation in immunodeficient or immunosuppressed patients, which can result in a lymphoma.
The triggering of EBV reactivation is poorly understood. It has however been clearly demonstrated that the viral ZEBRA protein (BamHI Z EBV replication activator), also called Zta, plays a role well upstream of this reactivation (George Miller, 1989 The Yale Journal of Biology and Medicine, 62: 205-213, El-Guindy et al., PLos Pathogens 2010, 6(8): e1001054). The ZEBRA protein, encoded by the BZLF-1 gene, is a transcription factor of the basic leucine-zipper family which plays an essential role in the transcription of the viral genes but also in the activation of virus replication. The ZEBRA protein is responsible for the passage of EBV from its latent phase to its lytic phase. The protein comprises three distinct functional domains: a transactivation domain, a DNA binding domain and a dimerization domain (Petosa et al., Molecular Cell 2006, 21(4): 565-572). This last domain, called ZANK domain (ZEBRA ANKryn-like Region) not only allows the ZEBRA protein to homodimerize but also to bind the p53 and NF-kB proteins (Dreyfus et al. Virology Journal 2011, 8:422). The interaction of the ZEBRA protein with the p53 and NF-kB proteins could play a role in the immunopathology and the viral carcinogenesis of the B lymphocytes induced by the EBV and other cell lines transitionally infected with the EBV.
The overexpression of the ZEBRA protein in cell lines of lymphocytes leads to the formation of ZEBRA-ZAP or ZEBRA-RACK1 protein dimers capable of modulating the transactivating activity of the ZEBRA protein or of disrupting the transduction of the protein kinase C-dependant signal respectively (Katz et al., PNAS, 1992, 89, 378-382; Tardif et al., JBC, 2002, 277(27), 24148-24154). However, these protein dimers have a purely intracellular location, either in the cytosol or in the nucleus of the cells overexpressing the ZEBRA protein; these dimers are absent from the culture medium. Access to these dimers is only possible following lysis of the cells, making their antibody assay indirect.
The study of pathologies in connection with EBV reactivation has made possible the identification of biological markers allowing the prognosis or diagnosis of lymphoproliferations in patients. Among these markers, ZEBRA constitutes a marker relevant to EBV reactivation.
Biologically, EBV reactivation has been characterized by the titration of anti-ZEBRA antibodies in patients who are carrying Hodgkin's disease (Drouet et al., J. Med. Virol. 1999, 57(4):383-9). The detection of anti-ZEBRA antibodies also constitutes a prognostic marker in patients suffering from nasopharyngeal carcinoma (Tedeschi et al., Clinical and Vaccine Immunology 2007, 14(4): 435-441/Dardari et al., J. Clin. Virol. 2008, 41(2): 96-103). Detection of the ZEBRA antigen has also served as a diagnostic marker of PTLD in patients who have received transplants. However, its measurement requires the performance of biopsies on which immunohistochemical techniques are carried out (Oertel et al., B. J. Haematology 2002, 118:1120-1123). Recourse to such techniques can only be envisaged in cases where the lymphoproliferation is located at a precise site in the organism and not diffused through different tissues.
Molecular biology techniques have made possible the emergence of commercial in vitro diagnostic tests for measuring the viral load of EBV. This measurement does not in itself constitute a marker for the diagnosis of PTLD but constitutes a diagnostic indicator informing the practitioner of the need to use or not use antivirals. However, the techniques used are not very standardized and vary from one analysis centre to another (Preiksaitis et al., Am. J. Transplant. 2009, 9(2): 269-279). In addition, work by Oertel shows that measurement of the viral load is not a good predictive marker of PTLDs (Oertel et al., 2006, Ann. Haematol., 85(7): 478-484).
As a result a real need exists for the identification of diagnostic markers of EBV reactivation which, when associated with other biological markers which are already known, allow the prognosis or diagnosis of lymphoproliferative episodes, whether a first episode or also a relapse.